Human macrophage antigen

ABSTRACT

The present invention provides a polynucleotide which identifies and encodes a novel human macrophage antigen (TMAH). The invention provides for genetically engineered expression vectors and host cells comprising the nucleic acid sequence encoding TMAH. The invention also provides for the use of substantially purified TMAH and its agonists, antibodies, antagonists or inhibitors in pharmaceutical compositions for treatment of diseases associated with expression of TMAH. The invention also describes diagnostic assays which utilize the polynucleotide to hybridize with the genomic sequence or transcripts encoding TMAH and anti-TMAH antibodies which specifically bind to TMAH.

FIELD OF THE INVENTION

The present invention relates to nucleic acid and amino acid sequencesof a novel human macrophage antigen which shares the featurescharacterizing hemopoietic-specific early response proteins and calciumdependent lectins and to the use of these sequences in the diagnosis,study, prevention and treatment of disease.

BACKGROUND OF THE INVENTION

The mouse hemopoietic-specific early response protein, A1, has beenstudied by Lin et al (1993; J Immunol 151:1979-88) who first reported onits relationship to Bcl-2 (B-cell leukemia/lymphoma 2)and Mcl-1. A1belongs to a gene family that has been characterized in humans, mice andchickens (Craig R. W. (1995) Semin Cancer Biol 6:35-43) and includesBfl-1, Bcl-2, Bcl-x, Bcl-xL, bax, and mcl-1. These genes regulate cellviability; in some cases, preventing toxicity to antibiotics andanticancer drugs (Minn A. J. et al (1995) Blood 86:1903-1910), and inothers, governing the apoptosis necessary for tissue differentiation andorganismal development. The exact mechanism by which A1, Bcl-2 andrelated genes promote cell survival is not known; however, they mayfunction through the inhibition of cysteine proteases.

The coding region of the murine A1 (Lin et al, supra) consists of 648nucleotides which encode 216 amino acids. The open reading frame (T₄₇₄)and the 3' untranslated region (T₇₁₅) each contain a TACAAA motif whichis found in many immediate early genes and may be essential forinduction. The deduced protein has a predicted molecular weight of20,024, an isoelectric point of 5.05, and a potential glycosylation siteat N₁₂₈. The absence of a secretion signal led Lin et al (supra) tosuggest that A1 might be an intracellular rather than a secretedprotein.

Expression of A1 has been detected in bone marrow, spleen and thymus andis induced by GM-CSF (granulocyte-macrophage colony stimulating factor)in several hematopoietic cell lineages, including T-helper lymphocytes,macrophages and neutrophils, and in myeloid cell lines induced todifferentiate by IL-3. A1 is induced in a macrophage tumor cell line bylipopolysaccharide. The protein synthesis inhibitor cycloheximide worksas an agonist to induce the long-term expression of A1, a featurepreviously reported for other early response genes (Lin, supra). Neitherserum nor the cytokines, interleukin (IL)-1 alpha or IL-6, induce A1expression.

Calcium dependent or C-type lectin receptors are widely expressed incells of the immune system. Their characteristic features include: 1) acytosolic amino terminus containing at least one potential tyrosinephosphorylation site which may be involved in signal transduction andseveral prolines which may prevent steric interference between thecytosolic and membrane spanning domains, 2) a short, approximately 20,hydrophobic amino acid transmembrane domain, and 3) a series of cysteineresidues which appear to function as an extracellular carbohydraterecognition domain (Speiss M. (1990) Biochem 29:10009-18). When theextracellular carbohydrate binding domain is separated from the membranespanning domain by protease activity, it maintains both its structuraland functional integrity. As described below, macrophage C-type lectinreceptors perform a variety of functions in the recognition anddestruction of foreign cells.

Diseases or Activities Associated with A1 or C-lectin Family Genes

Alterations or aberrations in A1 family gene expression are known toresult in premature cell death or in cancer. The best known gene in thisfamily, Bcl-2, is a proto-oncogene associated with human follicularlymphoma (Tsujimoto Y. and Croce C. M. (1986) Proc Nat Acad Sci83:5214), malignant melanomas, and solid tumors such as carcinomas ofthe lung, prostate and nasopharynx (Cerroni L. (1995) Am J.Dermatopathol 17:7-11). In patients with AIDS, there is also a highcorrelation between Epstein-Barr virus (EBV) and primary brainlymphomas. The virus's latent membrane protein 1 has been reported totransactivate the Bcl-2 gene and in one study, both genes were expressedin 10 out of 11 cases of AIDS-related primary brain lymphoma. Expressionof Bcl-2 is far less common in systemic lymphomas and cutaneous B or Tcell lymphomas (Garatti S. A. et al (1995) Recent Results Cancer Res139: 249-261).

Estrogen also increases Bcl-2 expression promoting chemotherapeutic drugresistance in an estrogen-responsive human breast cancer cell line(Teizeira C. et al (1995) Cancer Res 55:3902-3907). Bcl-2 is normallyexpressed in the epithelial regenerative compartment or the basal cryptsof the colon and small intestine. Overexpression of Bcl-2 was not seenin inflammatory gastrointestinal conditions such as ulcerative colitis,Crohn's disease, or hamartomatous polyps; but it was common inhyperplastic colonic polyps and in the majority of dysplastic lesions,adenomas and adenocarcinomas. The presence of excess Bcl-2 in tissuesurrounding the lesions suggests that the neoplasias arose from tissuein which earlier conversion to abnormal Bcl-2 expression occurred(Bronner M. P. et al (1995) Am J. Pathol 146:20-26).

Some of the other A1 family genes are now being characterized. Clinicalstudies on the gene, Bfl-1 (Choi S. S. et al (1995) Oncogene 11:1693-98)isolated from human fetal liver and highly expressed in bone marrow haveshown a correlation between expression of Bfl-1 and stomach cancer. Thestudies suggest that Bfl-1 may promote the survival of stomach cancercells by preventing apoptosis. Expression of Bci-XL dramatically reducescytotoxicity to antibiotics and chemotherapeutics such as bleomycin,cysplatin, hygromycin, and vincristine, (Minn, supra; Newcomb E. W.(1995) Leuk Lymphoma 17:211-221).

Silvestris F. et al (1995; Ann Ital Med Int 10:7-13) showed that A1family genes are involved in autoimmune conditions such as lupuserythematosus and degenerative neuropathies such as Alzheimer's disease;and Erlacher et al (1995; J. Rheumatol 22:926-931) reported on theincreased expression of Bcl-2 in chondrocytes adjacent to osteoarthriticdefects. Other studies suggest that inducing the expression of A1 familygenes may serve to rescue neurons from programmed cell death due tohypoxia (Shimizu et al (1995) Nature 374:811-816) or cerebral ischemicstroke (Linnik M. D. et al (1995; Stroke 26:1670-74).

Unique C type lectin receptors may direct the macrophages to abnormal ordiseased cells where they specifically interact with surface antigens.For example, Klebsiella pneumoniae serotypes displaying certain surfacemannose polysaccharide sequences bind to and are subsequentlyinternalized and destroyed by macrophages (Athamna A. et al (1991)Infect Immun 59:1673-1682). Similarly, the Tn Ag of a well-known humancarcinoma-associated epitope (Suzki N. et al (1996) J Immunol156:128-135) is recognized by a human macrophage C-type lectin. Bindingof macrophages to mastocytoma cells occurs through theGal/GalNAc-specific macrophage lectin and activates the tumor cellkilling mechanism (Oda S. et al (1989) J Biochem 105:1040-1043).

Some diseases are due to defects in the recognition of what is foreignas mediated via the macrophage lectin receptor, and some conditions suchas graft and transplant rejection and pathogen colonization of hostmacrophages derive from normal, yet undesirable, functioning ofmacrophages. For example, in rat cardiac allografts, expression ofmacrophage cell-surface lectins were linked to chronic rejection. Inthis case the lectin served as a possible mediator of macrophageinfiltration (Russel M. E. et al (1994) J Clin Invest 94: 722-730).Also, the attachment of such pathogens as Mycobacteria tuberculosis viamannose-specific lectin receptors expressed on the macrophages (GoswamiS. et al (1994) FEBS Lett 355:183-186) is the preliminary step inpathogenesis.

Macrophage antigens/lectins clearly play an important role in therecognition and destruction of foreign and diseased cells. The selectivemodulation of the expression and specificity of a novel human macrophageantigen may allow the successful management of diseases related tomacrophage function, allowing natural cytolysis via specific targetingto infected host cells or tumors or preventing graft rejection andpathogen colonization.

SUMMARY

The present invention discloses a novel human macrophage antigen,hereinafter referred to as TMAH and characterized as having structuralhomology to both mammalian A1 and the C-type lectins. Accordingly, theinvention features a substantially purified TMAH, as shown in the aminoacid sequence of SEQ ID NO:1. This sequence has a distinctive A₄₂ -A₆₁transmembrane motif and an extracellular domain, from residue S₆₂ toresidue I₂₇₂, which appears to bind to molecules such as lectins,carbohydrates or glycoproteins.

One aspect of the invention features isolated and substantially purifiedpolynucleotides which encode TMAH. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2. In addition,the invention features polynucleotide sequences that hybridize understringent conditions to SEQ ID NO:2.

The invention further relates to the nucleic acid sequence encodingTMAH, oligonucleotides, peptide nucleic acids (PNA), fragments, portionsor antisense molecules thereof. The invention also provides for the useof antisense molecules to disrupt the expression of the genomic sequenceencoding TMAH particularly in tissues involved in the development oflymphomas or carcinomas. The present invention relates, in part, to theinclusion of the nucleic acid sequence encoding TMAH in an expressionvector which can be used to transform host cells or organisms.

The present invention also relates to a method for producing TMAH or afragment thereof, antibodies which bind specifically to TMAH, and apharmaceutical composition comprising a substantially purified fragmentof TMAH in conjunction with a suitable pharmaceutical carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of the human macrophage antigenproduced using MacDNAsis™ software (Hitachi Software Engineering CoLtd).

FIGS. 2A and 2B shows the amino acid sequence alignments among humanmacrophage antigen (SEQ ID NO:1), mouse A1 (GI 239274; SEQ ID NO:3),human bcl-2 α (GI 179367; SEQ ID NO:4), and human mcl-1 (GI 34678; SEQID NO: 5) produced using the multisequence alignment program of DNAStar™software (DNAStar Inc, Madison Wis.).

FIGS. 3A and 3B show the amino acid sequence alignments among humanmacrophage antigen (SEQ ID NO:1), Type II integral membrane proteins (GI35061, SEQ ID NO:6; GI 35059,SEQ ID NO:7; and GI35057, SEQ ID NO:8) andCD94 (GI 1098617, SEQ ID NO:9).

FIG. 4 shows the hydrophobicity plot for human macrophage antigen, SEQID NO:1, generated using MacDNAsis software; the X axis reflects aminoacid position, and the negative Y axis, hydrophobicity.

DESCRIPTION OF THE INVENTION

Definitions

Definitions

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,amino acid sequence as used herein refers to peptide or proteinsequence.

"Peptide nucleic acid" as used herein refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary (template) strand of nucleic acid (Nielsen P. E. etal (1993) Anticancer Drug Des 8:53-63).

A "deletion" is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, are absent.

An "insertion" or "addition" is that change in a nucleotide or aminoacid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to thenaturally occurring TMAH.

A "substitution" results from the replacement of one or more nucleotidesor amino acids by different nucleotides or amino acids, respectively.

As used herein, TMAH refers to the amino acid sequence of substantiallypurified TMAH obtained from any species, particularly mammalian,including bovine, ovine, porcine, murine, equine, and preferably human,from any source whether natural, synthetic, semi-synthetic orrecombinant.

A "variant" of TMAH is defined as an amino acid sequence that isdifferent by one or more amino acid substitutions. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, eg, replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,eg, replacement of a glycine with a tryptophan. Similar minor variationsmay also include amino acid deletions or insertions, or both. Guidancein determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNAStar software.

The term "biologically active" refers to a TMAH having structural,regulatory or biochemical functions of a naturally occurring TMAH.Likewise, "immunologically active" defines the capability of thenatural, recombinant or synthetic TMAH, or any oligopeptide thereof, toinduce a specific immune response in appropriate animals or cells and tobind with specific antibodies.

The term "derivative" as used herein refers to the chemical modificationof a nucleic acid encoding TMAH or the encoded TMAH. Illustrative ofsuch modifications would be replacement of hydrogen by an alkyl, acyl,or amino group. A nucleic acid derivative would encode a polypeptidewhich retains essential biological characteristics of natural TMAH.

As used herein, the term "substantially purified" refers to molecules,either nucleic or amino acid sequences, that are removed from theirnatural environment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

The term "hybridization" as used herein shall include "any process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing" (Coombs J. (1994) Dictionary of Biotechnology, StocktonPress, New York N.Y.). Amplification is defined as the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction technologies well known in the art(Dieffenbach C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview N.Y.).

"Stringency" typically occurs in a range from about Tm-5° C. (5° C.below the Tm of the probe)to about 20° C. to 25° C. below Tm. As will beunderstood by those of skill in the art, a stringency hybridization canbe used to identify or detect identical polynucleotide sequences or toidentify or detect similar or related polynucleotide sequences.

Description

The present invention relates to a novel human macrophage antigeninitially identified as Incyte Clone 513418 (SEQ ID NO:2), using acomputer-generated search for amino acid sequence alignments. Theinvention also relates to the use of the disclosed nucleic acid andamino acid sequences in the study, diagnosis, prevention and treatmentof diseases associated with expression of these sequences. Although thehuman macrophage antigen encoding nucleotide sequence was identifiedamong the partial cDNAs from a macrophage library (MPHGNOT03), thenaturally occurring expression is not necessarily limited tomacrophages.

The present invention also encompasses human macrophage antigenvariants. A preferred variant is one having at least 80% amino acidsequence similarity to the TMAH of SEQ ID NO:1, a more preferred variantis one having at least 90% sequence similarity to SEQ ID NO:1, and amost preferred variant is one having at least 95% sequence similarity toSEQ ID NO:1.

The nucleic acid sequence, SEQ ID NO:2; disclosed herein encodes theamino acid sequence, SEQ ID NO:1, for the novel human macrophageantigen, designated herein as TMAH. The present invention is based, inpart, on the amino acid homology among TMAH, mouse A1 (GI 239274;),human bcl-2 α (GI 179367;), and human mcl-1 (GI 34678;) as shown in FIG.2, and among TMAH, the Type II integral membrane proteins (GI 35061, SEQID NO:6; GI 35059, SEQ ID NO:7; and GI35057, SEQ ID NO:8) and CD94 (GI1098617, SEQ ID NO:9) as shown in FIG. 3. TMAH has 20% amino acidsequence identity to each of these groups and is a transmembrane proteinwith an isoelectric point of 8.79. The homology which TMAH shares withthe other A1 family members is spread over the length of the amino acidsequence and includes conserved residues at F₂₇, P₃₅, R₁₁₉, W₁₃₉, F₁₄₆,and W₂₁₄ (FIG. 2). In addition, 23 serine and nine tyrosine residues arelocated in either the cytoplasmic or the extracellular portion of TMAHand present potential sites for phosphorylation. The transmembranespanning residues of TMAH, from R₄₀ to A₆₁ inclusive, closely matchesthat of CD94 (Chang C. et al (1995) Eur J Immunol 25:2433-2437). Theextracellular carbohydrate binding domain of TMAH contains the disulfideforming residues C₁₃₀, C₁₃₃, C₁₄₄, C₁₆₁, C₂₂₇, C₂₄₀ and C₂₄₈ (FIG. 3).TMAH is 272 amino acids long, has a potential cytoplasmicphosphorylation site at Y₇, and has seven potential glycosylation sites,N₁₄, N₈₀, N₈₈, N₉₈, N₁₀₅, N₁₆₅, and N₂₁₀ (FIG. 2).

The TMAH Coding Sequences

The nucleic acid and deduced amino acid sequences of TMAH are shown inFIGS. 1A and 1B. In accordance with the invention, any nucleic acidsequence which encodes TMAH can be used to generate recombinantmolecules which express TMAH. In a specific embodiment described herein,a partial sequence encoding TMAH was first isolated as Incyte Clone513418 from a macrophage cDNA library (MPHGNOT03).

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of TMAH-encodingnucleotide sequences, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene may be produced. Theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequenceencoding naturally occurring TMAH, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode TMAH and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring sequence under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding TMAH or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding TMAH and itsderivatives without altering the encoded amino acid sequences includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

It is now possible to produce a DNA sequence, or portions thereof,encoding TMAH and its derivatives entirely by synthetic chemistry, afterwhich the synthetic gene may be inserted into any of the many availableDNA vectors and cell systems using reagents that are well known in theart at the time of the filing of this application. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingTMAH or any portion thereof.

Also included within the scope of the present invention arepolynucleotide sequences that are capable of hybridizing to thenucleotide sequence of FIGS. 1A and 1B under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Berger and Kimmel (1987, Guide to Molecular Cloning Techniques,Methods in Enzymology, Vol 152, Academic Press, San Diego Calif.)incorporated herein by reference, and may be used at a defined"stringency".

Altered nucleic acid sequences encoding TMAH which may be used inaccordance with the invention include deletions, insertions orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent TMAH. The protein mayalso show deletions, insertions or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentTMAH. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of TMAH is retained. For example, negativelycharged amino acids include aspartic acid and glutamic acid; positivelycharged amino acids include lysine and arginine; and amino acids withuncharged polar head groups having similar hydrophilicity values includeleucine, isoleucine, valine; glycine, alanine; asparagine, glutamine;serine, threonine phenylalanine, and tyrosine.

Included within the scope of the present invention are alleles encodingTMAH. As used herein, an "allele" or "allelic sequence" is analternative form of the nucleic acid sequence encoding TMAH. Allelesresult from a mutation, ie, a change in the nucleic acid sequence, andgenerally produce altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions or substitutionsof amino acids. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing are well known in the art and employ suchenzymes as the Klenow fragment of DNA polymerase I, Sequenase® (USBiochemical Corp, Cleveland Ohio)), Taq polymerase (Perkin Elmer,Norwalk Conn.), thermostable T7 polymerase (Amersham, Chicago Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

Extending the Polynucleotide Sequence

The polynucleotide sequence encoding TMAH may be extended utilizingpartial nucleotide sequence and various methods known in the art todetect upstream sequences such as promoters and regulatory elements.Gobinda et al (1993; PCR Methods Applic 2:318-22) disclose"restriction-site" polymerase chain reaction (PCR) as a direct methodwhich uses universal primers to retrieve unknown sequence adjacent to aknown locus. First, genomic DNA is amplified in the presence of primerto a linker sequence and a primer specific to the known region. Theamplified sequences are subjected to a second round of PCR with the samelinker primer and another specific primer internal to the first one.Products of each round of PCR are transcribed with an appropriate RNApolymerase and sequenced using reverse transcriptase.

Inverse PCR can be used to amplify or extend sequences using divergentprimers based on a known region (Triglia T. et al (1988) Nucleic AcidsRes 16:8186). The primers may be designed using OLIGO® 4.06 PrimerAnalysis Software (1992; National Biosciences Inc, Plymouth Minn.), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Capture PCR (Lagerstrom M. et al (1991) PCR Methods Applic 1:111-19) isa method for PCR amplification of DNA fragments adjacent to a knownsequence in human and yeast artificial chromosome DNA. Capture PCR alsorequires multiple restriction enzyme digestions and ligations to placean engineered double-stranded sequence into an unknown portion of theDNA molecule before PCR.

Another method which may be used to retrieve unknown sequence is walkingPCR (Parker J. D. et al (1991) Nucleic Acids Res 19:3055-60), a methodfor targeted gene walking. Alternatively, PCR, nested primers,PromoterFinder™ (Clontech, Palo Alto Calif.) and PromoterFinderlibraries can be used to walk in genomic DNA. This process avoids theneed to screen libraries and is useful in finding intron/exon junctions.

Preferred libraries for screening for full length cDNAs are ones thathave been size-selected to include larger cDNAs. Also, random primedlibraries are preferred in that they will contain more sequences whichcontain the 5' and upstream regions of genes. A randomly primed librarymay be particularly useful if an oligo d(T) library does not yield afull-length cDNA. Genomic libraries are useful for extension into the 5'nontranslated regulatory region.

Capillary electrophoresis may be used to analyze either the size orconfirm the nucleotide sequence in sequencing or PCR products. Systemsfor rapid sequencing are available from Perkin Elmer, BeckmanInstruments (Fullerton Calif.), and other companies. Capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity is converted to electricalsignal using appropriate software (eg. Genotyper™ and SequenceNavigator™ from Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display is computercontrolled. Capillary electrophoresis is particularly suited to thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample. The reproducible sequencing of up to 350bp of M13 phage DNA in 30 min has been reported (Ruiz-Martinez M. C. etal (1993) Anal Chem 65:2851-8).

Expression of the Nucleotide Sequence

In accordance with the present invention, polynucleotide sequences whichencode TMAH, fragments of the polypeptide, fusion proteins or functionalequivalents thereof may be used in recombinant DNA molecules that directthe expression of TMAH in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequence,may be used to clone and express TMAH. As will be understood by those ofskill in the art, it may be advantageous to produce TMAH-encodingnucleotide sequences possessing non-naturally occurring codons. Codonspreferred by a particular prokaryotic or eukaryotic host (Murray E. etal (1989) Nuc Acids Res 17:477-508) can be selected, for example, toincrease the rate of TMAH expression or to produce recombinant RNAtranscripts having desirable properties, such as a longer half-life,than transcripts produced from naturally occurring sequence.

The nucleotide sequences of the present invention can be engineered inorder to alter a TMAH-encoding sequence for a variety of reasons,including but not limited to, alterations which modify the cloning,processing and/or expression of the gene product. For example, mutationsmay be introduced using techniques which are well known in the art, eg,site-directed mutagenesis to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, etc.

In another embodiment of the invention, a natural, modified orrecombinant TMAH-encoding sequence may be ligated to a heterologoussequence to encode a fusion protein. For example, for screening ofpeptide libraries for inhibitors of TMAH activity, it may be useful toencode a chimeric TMAH protein that is recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between a TMAH and the heterologous proteinsequence, so that the TMAH may be cleaved and substantially purifiedaway from the heterologous moiety.

In an alternate embodiment of the invention, the sequence encoding TMAHmay be synthesized, whole or in part, using chemical methods well knownin the art (see Caruthers M. H. et al (1980) Nuc Acids Res Symp Ser215-23, Horn T et al(1980) Nuc Acids Res Symp Ser 225-32, etc).Alternatively, the protein itself could be produced using chemicalmethods to synthesize a TMAH amino acid sequence, whole or in part. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge J. Y. et al (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer) in accordance with the instructionsprovided by the manufacturer.

The newly synthesized peptide can be substantially purified bypreparative high performance liquid chromatography (eg, Creighton (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co, NewYork N.Y.). The composition of the synthetic peptides may be confirmedby amino acid analysis or sequencing (eg, the Edman degradationprocedure; Creighton, supra). Additionally the amino acid sequence ofTMAH, or any part thereof, may be altered during direct synthesis and/orcombined using chemical methods with sequences from other proteins, orany part thereof, to produce a variant polypeptide.

Expression Systems

In order to express a biologically active TMAH, the nucleotide sequenceencoding TMAH or its functional equivalent, is inserted into anappropriate expression vector, ie, a vector which contains the necessaryelements for the transcription and translation of the inserted codingsequence.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a TMAH-encoding sequence andappropriate transcriptional or translational controls. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination or genetic recombination. Such techniques aredescribed in Sambrook et al (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview N.Y. and Ausubel F. M. et al(1989) Current Protocols in Molecular Biology, John Wiley & Sons, NewYork N.Y.

A variety of expression vector/host systems may be utilized to containand express a TMAH-encoding sequence. These include but are not limitedto microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (eg, baculovirus); plant cell systemstransfected with virus expression vectors (eg, cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (eg, Ti or pBR322 plasmid); or animal cell systems.

The "control elements" or "regulatory sequences" of these systems varyin their strength and specificities and are those nontranslated regionsof the vector, enhancers, promoters, and 3' untranslated regions, whichinteract with host cellular proteins to carry out transcription andtranslation. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla Calif.)or pSport1 (Gibco BRL) and ptrp-lac hybrids and the like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (eg, heatshock, RUBISCO; and storage protein genes) or from plant viruses (eg,viral promoters or leader sequences) may be cloned into the vector. Inmammalian cell systems, promoters from the mammalian genes or frommammalian viruses are most appropriate. If it is necessary to generate acell line that contains multiple copies of the sequence encoding TMAH,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for TMAH. For example, when largequantities of TMAH are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be desirable. Such vectors include, but are not limited to,the multifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding TMAH may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster (1989) J BiolChem 264:5503-5509); and the like. pGEX vectors (Promega, Madison Wis.)may also be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems are designed to includeheparin, thrombin or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase and PGH may be used. For reviews, see Ausubel et al (supra) andGrant et al (1987) Methods in Enzymology 153:516-544.

In cases where plant expression vectors are used, the expression of asequence encoding TMAH may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMV(Brisson et al (1984) Nature 310:511-514) may be used alone or incombination with the omega leader sequence from TMV (Takamatsu et al(1987) EMBO J 6:307-311). Alternatively, plant promoters such as thesmall subunit of RUBISCO (Coruzzi et al (1984) EMBO J 3:1671-1680;Broglie et al (1984) Science 224:838-843); or heat shock promoters(Winter J. and Sinibaldi R. M. (1991) Results Probl Cell Differ17:85-105) may be used. These constructs can be introduced into plantcells by direct DNA transformation or pathogen-mediated transfection.For reviews of such techniques, see Hobbs S or Murry L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill New YorkN.Y., pp 191-196 or Weissbach and Weissbach (1988) Methods for PlantMolecular Biology, Academic Press, New York N.Y., pp 421-463.

An alternative expression system which could be used to express TMAH isan insect system. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequenceencoding TMAH may be cloned into a nonessential region of the virus,such as the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of the sequence encoding TMAH will renderthe polyhedrin gene inactive and produce recombinant virus lacking coatprotein coat. The recombinant viruses are then used to infect S.frugiperda cells or Trichoplusia larvae in which TMAH is expressed(Smith et al (1983) J Virol 46:584; Engelhard E. K. et al (1994) ProcNat Acad Sci 91:3224-7).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a sequence encoding TMAH may be ligated into an adenovirustranscription/ translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a nonessential E1 or E3 regionof the viral genome will result in a viable virus capable of expressingin infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci81:3655-59). In addition, transcription enhancers, such as the roussarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Specific initiation signals may also be required for efficienttranslation of a sequence encoding TMAH. These signals include the ATGinitiation codon and adjacent sequences. In cases where the sequenceencoding TMAH, its initiation codon and upstream sequences are insertedinto the most appropriate expression vector, no additional translationalcontrol signals may be needed. However, in cases where only codingsequence, or a portion thereof, is inserted, exogenous transcriptionalcontrol signals including the ATG initiation codon must be provided.Furthermore, the initiation codon must be in the correct reading frameto ensure transcription of the entire insert. Exogenous transcriptionalelements and initiation codons can be of various origins, both naturaland synthetic. The efficiency of expression may be enhanced by theinclusion of enhancers appropriate to the cell system in use (Scharf D.et al (1994) Results Probl Cell Differ 20:125-62; Bittner et al (1987)Methods in Enzymol 153:516-544).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etchave specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressTMAH may be transformed using expression vectors which contain viralorigins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellsmay be allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clumps of stably transformed cells can be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler M. et al (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy I. et al (1980) Cell 22:817-23) geneswhich can be employed in tk- or aprt-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler M. et al (1980) Proc Natl Acad Sci 77:3567-70);npt, which confers resistance to the aminoglycosides neomycin and G-418(Colbere-Garapin F. et al (1981) J Mol Biol 150:1-14) and als or pat,which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman S. C. and R. C. Mulligan(1988) Proc Natl Acad Sci 85:8047-51). Recently, the use of visiblemarkers has gained popularity with such markers as anthocyanins, βglucuronidase and its substrate, GUS, and luciferase and its substrate,luciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes C. A. et al (1995)Methods Mol Biol 55:121-131).

Identification of Transformants Containing the Polynucleotide Sequence

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression shouldbe confirmed. For example, if the sequence encoding TMAH is insertedwithin a marker gene sequence, recombinant cells containing the sequenceencoding TMAH can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with the sequenceencoding TMAH under the control of a single promoter. Expression of themarker gene in response to induction or selection usually indicatesexpression of the tandem sequence as well.

Alternatively, host cells which contain the coding sequence for TMAH andexpress TMAH may be identified by a variety of procedures known to thoseof skill in the art. These procedures include, but are not limited to,DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassaytechniques which include membrane, solution, or chip based technologiesfor the detection and/or quantification of the nucleic acid or protein.

The presence of the polynucleotide sequence encoding TMAH can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of the sequence encoding TMAH. Nucleicacid amplification based assays involve the use of oligonucleotides oroligomers based on the nucleic acid sequence to detect transformantscontaining DNA or RNA encoding TMAH. As used herein "oligonucleotides"or "oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides which can beused as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofTMAH, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson TMAH is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton R.et al (1990, Serological Methods, a Laboratory Manual, APS Press, StPaul Minn.) and Maddox D. E. et al (1983, J Exp Med 158:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting related sequences include oligolabeling, nick translation,end-labeling or PCR amplification using a labeled nucleotide.Alternatively, the TMAH-encoding sequence, or any portion of it, may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also,recombinant immunoglobulins may be produced as shown in U.S. Pat. No.4,816,567 incorporated herein by reference.

Purification of TMAH

Host cells transformed with a nucleotide sequence encoding TMAH may becultured under conditions suitable for the expression and recovery ofthe encoded protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing TMAH-encodingsequence can be designed with signal sequences which direct secretion ofTMAH through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may join the sequence encoding TMAH tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins (Kroll D. J. et al (1993) DNA Cell Biol12:441-53; of discussion of vectors infra containing fusion proteins).

TMAH may also be expressed as a recombinant protein with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequences such as Factor XAor enterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and TMAH is useful to facilitate purification. One suchexpression vector provides for expression of a fusion protein comprisingthe sequence encoding TMAH and nucleic acid sequence encoding 6histidine residues followed by thioredoxin and an enterokinase cleavagesite. The histidine residues facilitate purification while theenterokinase cleavage site provides a means for purifying TMAH from thefusion protein.

In addition to recombinant production, fragments of TMAH may be producedby direct peptide synthesis using solid-phase techniques (cf Stewart etal (1969) Solid-Phase Peptide Synthesis, W. H. Freeman Co, SanFrancisco; Merrifield J. (1963) J Am Chem Soc 85:2149-2154). In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster CityCalif.) in accordance with the instructions provided by themanufacturer. Various fragments of TMAH may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

Uses of TMAH

The rationale for use of the nucleotide and peptide sequences disclosedherein is based on the chemical and structural homology among the novelhuman macrophage antigen and the A1 (GI 293273; Lin et al. (1993) JImmunol 151:1979-88) and C-type lectin receptor (GI 350597, GI 35059,GI35061; Houchins J. P. et al (1991) J Exp Med 173:1017-20) genefamilies. Furthermore, since expression of the A1 family genes isusually associated with cells of hematopoeitic origin, it appears thatvariants of these genes will be found in similar cells or tissues wherethey will carry similar functions.

The similarity of TMAH to members of the A1 gene family suggests thatenhancing the expression of TMAH would promote cell survival. Forexample, providing TMAH to joint chondrocytes may result in decreasedapoptosis and slow the progressive degeneration associated with theonset of osteoarthritis. Similarly, inducing the expression of TMAH mayserve to slow other immune or senescent responses such as the rescue ofneurons from programmed cell death due to hypoxia or cerebral ischemicstroke. Thus the expression of TMAH may promote cell survival in casesof immune or senescent diseases including, but not limited to, AIDS,Alzheimer's disease, arthritis, graft and transplant rejection, lupuserythematosis, and myasthenia gravis.

In those situations where the induction of apoptosis is desirable, cellscould be transfected with antisense sequences of the gene encoding TMAHor provided with inhibitors of TMAH. Such diseases include cancers suchas lymphoma, malignant melanomas, and carcinomas of the breast,intestine, lung, nasopharynx, and prostate.

TMAH Antibodies

TMAH-specific antibodies are useful for the diagnosis and treatment ofconditions and diseases associated with expression of TMAH. Suchantibodies include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fab fragments and fragments produced by a Fabexpression library. Neutralizing antibodies, ie, those which inhibitdimer formation, are especially preferred for diagnostics andtherapeutics.

TMAH for antibody induction does not require biological activity;however, the protein fragment, or oligopeptide must be antigenic.Peptides used to induce specific antibodies may have an amino acidsequence consisting of at least five amino acids, preferably at least 10amino acids. Preferably, they should mimic a portion of the amino acidsequence of the natural protein and may contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofTMAH amino acids may be fused with those of another protein such askeyhole limpet hemocyanin and antibody produced against the chimericmolecule. Procedures well known in the art can be used for theproduction of antibodies to TMAH.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, etc may be immunized by injection with TMAH or anyportion, fragment or oligopeptide which retains immunogenic properties.Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include but are not limited toFreund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants.

Monoclonal antibodies to TMAH may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include but are not limited to the hybridomatechnique originally described by Koehler and Milstein (1975 Nature256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983)Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030)and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al (1984) Proc Natl AcadSci 81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda etal (1985) Nature 314:452-454). Alternatively, techniques described forthe production of single chain antibodies (U.S. Pat. No. 4,946,778) canbe adapted to produce TMAH-specific single chain antibodies.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G.and Milstein C. (1991; Nature 349:293-299).

Antibody fragments which contain specific binding sites for TMAH mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse W. D. et al (1989) Science 256:1275-1281).

A variety of protocols for competitive binding or immunoradiometricassays using either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the formation of complexes between TMAH and its specificantibody and the measurement of complex formation. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo noninterfering epitopes on a specific TMAH protein is preferred, buta competitive binding assay may also be employed. These assays aredescribed in Maddox D. E. et al (1983, J Exp Med 158:1211).

Diagnostic Assays Using TMAH Specific Antibodies

Particular TMAH antibodies are useful for the diagnosis of conditions ordiseases characterized by expression of TMAH or in assays to monitorpatients being treated with TMAH, its fragments, agonists or inhibitors.Diagnostic assays for TMAH include methods utilizing the antibody and alabel to detect TMAH in human body fluids or extracts of cells ortissues. The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a reporter molecule. A wide variety of reportermolecules are known, several of which were described above.

A variety of protocols for measuring TMAH, using either polyclonal ormonoclonal antibodies specific for the respective protein are known inthe art. Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on TMAH is preferred, but acompetitive binding assay may be employed. These assays are described,among other places, in Maddox, D. E. et al (1983, J Exp Med 158:1211).

In order to provide a basis for diagnosis, normal or standard values forTMAH expression must be established. This is accomplished by combiningbody fluids or cell extracts taken from normal subjects, either animalor human, with antibody to TMAH under conditions suitable for complexformation which are well known in the art. The amount of standardcomplex formation may be quantified by comparing various artificialmembranes containing known quantities of TMAH with both control anddisease samples from biopsied tissues. Then, standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom subjects potentially affected by disease. Deviation betweenstandard and subject values establishes the presence of disease state.

Drug Screening

TMAH, its catalytic or immunogenic fragments or oligopeptides thereof,can be used for screening therapeutic compounds in any of a variety ofdrug screening techniques. The fragment employed in such a test may befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. The formation of binding complexes, betweenTMAH and the agent being tested, may be measured.

Another technique for drug screening which may be used for highthroughput screening of compounds having suitable binding affinity tothe TMAH is described in detail in "Determination of Amino Acid SequenceAntigenicity" by Geysen H. N., WO Application 84/03564, published onSep. 13, 1984, and incorporated herein by reference. In summary, largenumbers of different small peptide test compounds are synthesized on asolid substrate, such as plastic pins or some other surface. The peptidetest compounds are reacted with fragments of TMAH and washed. Bound TMAHis then detected by methods well known in the art. Substantiallypurified TMAH can also be coated directly onto plates for use in theaforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding TMAHspecifically compete with a test compound for binding TMAH. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with TMAH.

Uses of the Polynucleotide Encoding TMAH

A polynucleotide sequence encoding TMAH or any part thereof may be usedfor diagnostic and/or therapeutic purposes. For diagnostic purposes, thesequence encoding TMAH of this invention may be used to detect andquantitate gene expression in biopsied tissues in which expression ofTMAH may be implicated. The diagnostic assay is useful to distinguishbetween absence, presence, and excess expression of TMAH and to monitorregulation of TMAH levels during therapeutic intervention. Included inthe scope of the invention are oligonucleotide sequences, antisense RNAand DNA molecules, and PNAs.

Another aspect of the subject invention is to provide for hybridizationor PCR probes which are capable of detecting polynucleotide sequences,including genomic sequences, encoding TMAH or closely related molecules.The specificity of the probe, whether it is made from a highly specificregion, eg, 10 unique nucleotides in the 5' regulatory region, or a lessspecific region, eg, especially in the 3' region, and the stringency ofthe hybridization or amplification (maximal, high, intermediate or low)will determine whether the probe identifies only naturally occurringTMAH, alleles or related sequences.

Probes may also be used for the detection of related sequences andshould preferably contain at least 50% of the nucleotides from any ofthese TMAH-encoding sequences. The hybridization probes of the subjectinvention may be derived from the nucleotide sequence of SEQ ID NO:2 orfrom genomic sequence including promoter, enhancer elements and intronsof the naturally occurring sequence encoding TMAH. Hybridization probesmay be labeled by a variety of reporter groups, including radionuclidessuch as 32P or 35S, or enzymatic labels such as alkaline phosphatasecoupled to the probe via avidin/biotin coupling systems, and the like.

Other means for producing specific hybridization probes for DNAs includethe cloning of nucleic acid sequences encoding TMAH or TMAH derivativesinto vectors for the production of mRNA probes. Such vectors are knownin the art and are commercially available and may be used to synthesizeRNA probes in vitro by means of the addition of the appropriate RNApolymerase as T7 or SP6 RNA polymerase and the appropriate radioactivelylabeled nucleotides.

Diagnostic Use

Polynucleotide sequences encoding TMAH may be used for the diagnosis ofconditions or diseases with which the expression of TMAH is associated.For example, polynucleotide sequences encoding TMAH may be used inhybridization or PCR assays of fluids or tissues from biopsies to detectTMAH expression. The form of such qualitative or quantitative methodsmay include Southern or northern analysis, dot blot or othermembrane-based technologies; PCR technologies; dip stick, pin, chip andELISA technologies. All of these techniques are well known in the artand are the basis of many commercially available diagnostic kits.

The TMAH-encoding nucleotide sequences disclosed herein provide thebasis for assays that detect activation or induction associated withinflammation or disease. The nucleotide sequence may be labeled bymethods known in the art and added to a fluid or tissue sample from apatient under conditions suitable for the formation of hybridizationcomplexes. After an incubation period, the sample is washed with acompatible fluid which optionally contains a dye (or other labelrequiring a developer) if the nucleotide has been labeled with anenzyme. After the compatible fluid is rinsed off, the dye is quantitatedand compared with a standard. If the amount of dye in the biopsied orextracted sample is significantly elevated over that of a comparablecontrol sample, the nucleotide sequence has hybridized with nucleotidesequences in the sample, and the presence of elevated levels ofnucleotide sequences encoding TMAH in the sample indicates the presenceof the associated inflammation and/or disease.

Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regime in animal studies, in clinical trials, orin monitoring the treatment of an individual patient. In order toprovide a basis for the diagnosis of disease, a normal or standardprofile for TMAH expression must be established. This is accomplished bycombining body fluids or cell extracts taken from normal subjects,either animal or human, with TMAH, or a portion thereof, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fornormal subjects with a dilution series of TMAH run in the sameexperiment where a known amount of substantially purified TMAH is used.Standard values obtained from normal samples may be compared with valuesobtained from samples from patients affected by TMAH-associateddiseases. Deviation between standard and subject values establishes thepresence of disease.

Once disease is established, a therapeutic agent is administered and atreatment profile is generated. Such assays may be repeated on a regularbasis to evaluate whether the values in the profile progress toward orreturn to the normal or standard pattern. Successive treatment profilesmay be used to show the efficacy of treatment over a period of severaldays or several months.

PCR, may be used as described in U.S. Pat. Nos. 4,683,195 and 4,965,188provides additional uses for oligonucleotides based upon the sequenceencoding TMAH. Such oligomers are generally chemically synthesized, butthey may be generated enzymatically or produced from a recombinantsource. Oligomers generally comprise two nucleotide sequences, one withsense orientation (5'→3') and one with antisense (3'←5'), employed underoptimized conditions for identification of a specific gene or condition.The same two oligomers, nested sets of oligomers, or even a degeneratepool of oligomers may be employed under less stringent conditions fordetection and/or quantitation of closely related DNA or RNA sequences.

Additionally, methods which may be used to quantitate the expression ofa particular molecule include radiolabeling (Melby P. C. et al 1993 JImmunol Methods 159:235-44) or biotinylating (Duplaa C. et al 1993 AnalBiochem 229-36) nucleotides, coamplification of a control nucleic acid,and standard curves onto which the experimental results areinterpolated. Quantitation of multiple samples may be speeded up byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation. A definitive diagnosis of this typemay allow health professionals to begin aggressive treatment and preventfurther worsening of the condition. Similarly, further assays can beused to monitor the progress of a patient during treatment. Furthermore,the nucleotide sequences disclosed herein may be used in molecularbiology techniques that have not yet been developed, provided the newtechniques rely on properties of nucleotide sequences that are currentlyknown such as the triplet genetic code, specific base pair interactions,and the like.

Therapeutic Use

Based upon its homology to A1 family members and its expression profile,the polynucleotide encoding TMAH disclosed herein may be useful in thetreatment of genetic or immune system disorders such as asthma,arthritis, etc and inflammatory conditions.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisense ofthe sequence encoding TMAH. See, for example, the techniques describedin Sambrook et al (supra) and Ausubel et al (supra).

The polynucleotides comprising full length cDNA sequence and/or itsregulatory elements enable researchers to use the sequence encoding TMAHas an investigative tool in sense (Youssoufian H. and H. F. Lodish 1993Mol Cell Biol 13:98-104) or antisense (Eguchi et al (1991) Annu RevBiochem 60:631-652) regulation of gene function. Such technology is nowwell known in the art, and sense or antisense oligomers, or largerfragments, can be designed from various locations along the coding orcontrol regions.

Genes encoding TMAH can be turned off by transfecting a cell or tissuewith expression vectors which express high levels of a desired TMAHfragment. Such constructs can flood cells with untranslatable sense orantisense sequences. Even in the absence of integration into the DNA,such vectors may continue to transcribe RNA molecules until all copiesare disabled by endogenous nucleases. Transient expression may last fora month or more with a non-replicating vector (Mettler I, personalcommunication) and even longer if appropriate replication elements arepart of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA or PNA, to the control regionsof the sequence encoding TMAH, ie, the promoters, enhancers, andintrons. Oligonucleotides derived from the transcription initiationsite, eg, between -10 and +10 regions of the leader sequence, arepreferred. The antisense molecules may also be designed to blocktranslation of mRNA by preventing the transcript from binding toribosomes. Similarly, inhibition can be achieved using "triple helix"base-pairing methodology. Triple helix pairing compromises the abilityof the double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA were reviewed by Gee J. E. et al (In: HuberB. E. and B. I. Carr (1994) Molecular and Immunologic Approaches, FuturaPublishing Co, Mt Kisco N.Y.).

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Withinthe scope of the invention are engineered hammerhead motif ribozymemolecules that can specifically and efficiently catalyze endonucleolyticcleavage of the sequence encoding TMAH.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of RNA molecules. Theseinclude techniques for chemically synthesizing oligonucleotides such assolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding TMAH. Such DNA sequences may be incorporated into awide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, antisense cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine and wybutosine as well as acetyl-, methyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Methods for introducing vectors into cells or tissues include thosemethods discussed infra and which are equally suitable for in vivo, invitro and ex vivo therapy. For ex vivo therapy, vectors are introducedinto stem cells taken from the patient and clonally propagated forautologous transplant back into that same patient as presented in U.S.Pat. Nos. 5,399,493 and 5,437,994, disclosed herein by reference.Delivery by transfection and by liposome are quite well known in theart.

Furthermore, the nucleotide sequences encoding TMAH disclosed herein maybe used in molecular biology techniques that have not yet beendeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including but not limited to suchproperties as the triplet genetic code and specific base pairinteractions.

Detection and Mapping of Related Polynucleotide Sequences

The nucleic acid sequence encoding TMAH can also be used to generatehybridization probes for mapping the naturally occurring genomicsequence. The sequence may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Theseinclude in situ hybridization to chromosomal spreads, flow-sortedchromosomal preparations, or artificial chromosome constructions such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price C. M. (1993; Blood Rev 7:127-34) and Trask B. J.(1991; Trends Genet 7:149-54).

The technique of fluorescent in situ hybridization of chromosome spreadshas been described, among other places, in Verma et al (1988) HumanChromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y.Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofa the sequence encoding TMAH on a physical chromosomal map and aspecific disease (or predisposition to a specific disease) may helpdelimit the region of DNA associated with that genetic disease. Thenucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier or affectedindividuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers are invaluable in extending genetic maps. A recent example of anSTS based map of the human genome was recently published by theWhitehead-MIT Center for Genomic Research (Hudson T. J. et al (1995)Science 270:1945-1954). Often the placement of a gene on the chromosomeof another mammalian species such as mouse (Whitehead Institute/MITCenter for Genome Research, Genetic Map of the Mouse, Database Release10, Apr. 28, 1995) may reveal associated markers even if the number orarm of a particular human chromosome is not known. New sequences can beassigned to chromosomal arms, or parts thereof, by physical mapping.This provides valuable information to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once a disease or syndrome, such as ataxia telangiectasia(AT), has been crudely localized by genetic linkage to a particulargenomic region, for example, AT to 11q22-23 (Gatti et al (1988) Nature336:577-580), any sequences mapping to that area may representassociated or regulatory genes for further investigation. The nucleotidesequence of the subject invention may also be used to detect differencesin the chromosomal location due to translocation, inversion, etc. amongnormal, carrier or affected individuals.

Pharmaceutical Compositions

The present invention relates to pharmaceutical compositions which maycomprise nucleotides, proteins, antibodies, agonists, antagonists, orinhibitors, alone or in combination with at least one other agent, suchas stabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. Any of these molecules canbe administered to a patient alone, or in combination with other agents,drugs or hormones, in pharmaceutical compositions where it is mixed withexcipient(s) or pharmaceutically acceptable carriers. In one embodimentof the present invention, the pharmaceutically acceptable carrier ispharmaceutically inert.

Administration of Pharmaceutical Compositions

Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co,Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, ie, dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

Manufacture and Storage

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, eg, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in a acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of TMAH, such labeling wouldinclude amount, frequency and method of administration.

Therapeutically Effective Dose

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, eg, of neoplastic cells, or inanimal models, usually mice, rabbits, dogs, or pigs. The animal model isalso used to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

A therapeutically effective dose refers to that amount of protein or itsantibodies, antagonists, or inhibitors which ameliorate the symptoms orcondition. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, eg, ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state, eg, tumor size and location; age, weightand gender of the patient; diet, time and frequency of administration,drug combination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

It is contemplated, for example, that a therapeutically effective doseof TMAH fragment consisting of amino acid residues S₆₂ to I₂₇₂ can bedelivered in a suitable formulation to act as a biological spongeagainst microbes or to deliver another therapeutic molecule to cancerouscells or tissues.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I cDNA Library Construction

The peripheral blood sample was obtained from a normal 24 year old maleCaucasian. Human peripheral blood mononuclear cells were separated fromheparinized venous blood after centrifugation through Ficoll/Hypaque.The Ficoll/Hypaque buffy coat which contains peripheral bloodmononuclear cells was put into sterile Petri dishes and cultured forbetween 3 to 5 days in Dulbecco's minimum essential medium (DME)supplemented with 10% human serum. After the incubation period,macrophages mostly adhered to the plastic surface, whereas most othercell types, B cells and T cells, remained in solution. The DMEsupplemented with 10% human serum was decanted from the wells and washedwith phosphate buffered saline (PBS). Macrophages were released fromplastic surface by gently scraping the Petri dishes in PBS/1 mM EDTA.Macrophages were lysed immediately in buffer containing guanidiniumisothiocyanate.

Lysates were then loaded on a 5.7M CsCl cushion and ultracentrifuged ina SW28 swinging bucket rotor for 18 hours at 25,000 rpm at ambienttemperature. Total RNA was then ethanol precipitated, washed in 70%ethanol and resuspended in distilled water and DNAse for 15 minutes at37° C. The RNA was acid phenol extracted was acid phenol extracted andethanol precipitated. After being washed in 70% ethanol, thepolyadenylated RNA was isolated using Oligotex™ resin with sphericallatex particles (QIAGEN Inc., Chatsworth Calif.) and quantitated andfrozen at -80° C. The isolated poly A+ mRNA was sent to Stratagene (LaJolla, Calif.) for custom cDNA library construction.

Stratagene prepared the cDNA library using oligo d(T) priming where theprimer also contains a Not1 site for directional cloning. Syntheticadapter oligonucleotides were ligated onto the cDNA molecules enablingthem to be inserted into the Uni-ZAP™ vector system (Stratagene)followed by sizing of the cDNA on a Sephacryl S1000 column. This allowedhigh efficiency unidirectional (sense orientation) lambda libraryconstruction and the convenience of a plasmid system with blue/whitecolor selection to detect clones with cDNA insertions.

The quality of the cDNA library was screened using DNA probes, and then,the pBluescript® phagemid (Stratagene) was excised. This phagemid allowsthe use of a plasmid system for easy insert characterization,sequencing, site-directed mutagenesis, the creation of unidirectionaldeletions and expression of fusion polypeptides. Subsequently, thecustom-constructed library phage particles were infected into E. colihost strain XL1-Blue® (Stratagene). The high transformation efficiencyof this bacterial strain increases the probability that the cDNA librarycontains rare, under-represented clones. Alternative unidirectionalvectors include, but are not limited to, pcDNAI (Invitrogen, San DiegoCalif.) and pSHlox-1 (Novagen, Madison Wis.).

The cDNAs were sequenced by the method of Sanger F. and A. R. Coulson(1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200 (Hamilton,Reno Nev.) in combination with four Peltier Thermal Cyclers (PTC200 fromMJ Research, Watertown Mass.) and Applied Biosystems 377 or 373 DNASequencing Systems, and the reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT-670Sequence Analysis System. In this algorithm, Pattern SpecificationLanguage (TRW Inc, Los Angeles Calif.) was used to determine regions ofhomology. The three parameters that determine how the sequencecomparisons run were window size, window offset, and error tolerance.Using a combination of these three parameters, the DNA database wassearched for sequences containing regions of homology to the querysequence, and the appropriate sequences were scored with an initialvalue. Subsequently, these homologous regions were examined using dotmatrix homology plots to distinguish regions of homology from chancematches. Smith-Waterman alignments were used to display the results ofthe homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT™ 670 Sequence Analysis System in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul S.F. (1993) J. Mol Evol 36:290-300; Altschul, S. F. et al (1990) J. MolBiol 215:403-10), was used to search for local sequence alignments.BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs. BLAST is useful for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al. supra).

Analogous computer techniques using BLAST (Altschul S. F. 1993 and 1990,supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (Incyte,Palo Alto Calif.). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## and it takes into account both the degree of similarity betweentwo sequences and the length of the sequence match. For example, with aproduct score of 40, the match will be exact within a 1-2% error; and at70, the match will be exact. Homologous molecules are usually identifiedby selecting those which show product scores between 15 and 40, althoughlower scores may identify related molecules.

V Extension of the Sequence Encoding TMAH

The nucleic acid sequence of SEQ ID NO:2 is used to designoligo-nucleotide primers for extending a partial nucleotide sequence tofull length or for obtaining 5' sequence from genomic libraries. Oneprimer is synthesized to initiate extension in the antisense direction(XLR) and the other is synthesized to extend sequence in the sensedirection (XLF). Primers allow the extension of the known sequence"outward" generating amplicons containing new, unknown nucleotidesequence for the region of interest (U.S. patent application Ser. No.08/487,112, filed Jun. 7, 1995, specifically incorporated by reference).The initial primers are designed from the cDNA using OLIGO® 4.06 PrimerAnalysis Software (National Biosciences), or another appropriateprogram, to be 22-30 nucleotides in length, to have a GC content of 50%or more, and to anneal to the target sequence at temperatures about68°-72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; MJ Research,Watertown Mass.) and the following parameters:

    ______________________________________                                        Step 1       940° C. for 1 min (initial denaturation)                  Step 2       65° C. for 1 min                                          Step 3       68° C. for 6 min                                          Step 4       940° C. for 15 sec                                        Step 5       65° C. for 1 min                                          Step 6       68° C. for 7 min                                          Step 7       Repeat step 4-6 for 15 additional cycles                         Step 8       940 ° C. for 15 sec                                       Step 9       65° C. for 1 min                                          Step 10      68° C. for 7:15 min                                       Step 11      Repeat step 8-10 for 12 cycles                                   Step 12      72° C. C for 8 min                                        Step 13      40° C. (and holding)                                      ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products were selected and cut out of the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQuick™ (QIAGEN Inc). After recovery of the DNA, Klenow enzymewas used to trim single-stranded, nucleotide overhangs creating bluntends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1μl T4-DNA ligase (15 units) and 1μl T4 polynucleotidekinase are added, and the mixture is incubated at room temperature for2-3 hours or overnight at 16° C. Competent E. coli cells (in 40 μl ofappropriate media) are transformed with 3 μl of ligation mixture andcultured in 80 μl of SOC medium (Sambrook J. et al, supra). Afterincubation for one hour at 37° C., the whole transformation mixture isplated on Luria Bertani (LB)-agar (Sambrook J. et al, supra) containing2×Carb. The following day, several colonies are randomly picked fromeach plate and cultured in 150 μl of liquid LB/2×Carb medium placed inan individual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture is transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample is transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

    ______________________________________                                        Step 1     940° C. for 60 sec                                          Step 2     940° C. for 20 sec                                          Step 3     55° C. for 30 sec                                           Step 4     72° C. for 90 sec                                           Step 5     Repeat steps 2-4 for an additional 29 cycles                       Step 6     72° C. for 180 sec                                          Step 7     4° C. (and holding)                                         ______________________________________                                    

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs or mRNAs. Although the labeling of oligonucleotides,consisting of about 20 base-pairs, is specifically described,essentially the same procedure is used with larger cDNA fragments.Oligonucleotides are designed using state-of-the-art software such asOLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of eacholigomer and 250 mCi of γ-³² P! adenosine triphosphate (Amersham,Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®, Boston Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 super fine resin column (Pharmacia). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genomicDNA digested with one of the following endonucleases (Ase I, Bgl II, EcoRI, Pst I, Xba 1, or Pvu II; DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale Calif.) for several hours,hybridization patterns are compared visually.

VII Antisense Molecules

The sequence encoding TMAH, or any part thereof, is used to inhibit invivo or in vitro expression of naturally occurring sequence. Althoughuse of antisense oligonucleotides, comprising about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger cDNA fragments. An oligonucleotide complementary to the codingsequence of TMAH as shown in FIGS. 1A, 1B and 1C is used to inhibitexpression of naturally occurring sequence. The complementaryoligonucleotide is designed from the most unique 5' sequence as shown inFIGS. 1A, 1B and 1C and used either to inhibit transcription bypreventing promoter binding to the upstream nontranslated sequence ortranslation of an TMAH-encoding transcript by preventing the ribosomefrom binding. Using an appropriate portion of the leader and 5' sequenceof SEQ ID NO:2, an effective antisense oligonucleotide includes any15-20 nucleotides spanning the region which translates into the signalor early coding sequence of the polypeptide as shown in FIGS. 1A, 1B and1C.

VIII Expression of TMAH

Expression of the TMAH is accomplished by subcloning the cDNAs intoappropriate vectors and transfecting the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express TMAH in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Metand the subsequent 7 residues of β-galactosidase. Immediately followingthese eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transfected bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firstseven residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length TMAH. The signal sequence directs the secretion of TMAHinto the bacterial growth media which can be used directly in thefollowing assay for activity.

IX TMAH Activity

TMAH activity can be assayed in BHK cells seeded on a microscope slideand transiently transfected with the following plasmids: one whichcontains the nucleic acid sequence encoding TMAH and one which containstandemly arranged coding sequences for tumor necrosis factor alpha(TNF-α; which causes apoptosis) and B-galactosidase. The cells are fixedafter twelve hours and incubated in a buffer containing X-gal tovisualize B-galactosidase activity. Phase or interference contrastmicroscopy is used to examine the slides. Cells expressing only theplasmid with TNF-α display shrunken nuclei, intense blue staining andmembrane blebbling. Cells expressing both plasmids show nearly normalnuclei, intense blue staining, and nearly normal membranes, noblebbling. This techniques was adapted from Stanger BZ (1995; Cell81:513-523).

In the alternative, C-type lectin receptor activity may be assayed byfirst labeling the polypeptide with ¹²⁵ I Bolton-Hunter reagent (BoltonA. E. and Hunter W. M. (1973) Biochem J. 133:529). Candidate ligands(including lectins, polysaccharides, glycoproteins) previously arrayedin the wells of a 96 well plate are incubated with membrane fragmentscontaining the labeled TMAH. The plates are washed, assayed andquantitated spectrophotometrically for ligand:TMAH complex. Dataobtained using different concentrations of the candidate ligands areused to calculate the number, distribution, and association of TMAH withthe candidate ligands.

X Production of TMAH Specific Antibodies

TMAH substantially purified using PAGE electrophoresis (Sambrook, supra)is used to immunize rabbits and to produce antibodies using standardprotocols. The amino acid sequence translated from TMAH is analyzedusing DNAStar software (DNAStar Inc) to determine regions of highimmunogenicity and a corresponding oligopolypeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Analysis to select appropriate epitopes, such as those near theC-terminus or in hydrophilic regions (shown in FIGS. 4 and 5) isdescribed by Ausubel F. M. et al (supra).

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) byreaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS;Ausubel F. M. et al, supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring TMAH Using Specific Antibodies

Naturally occurring or recombinant TMAH is substantially purified byimmunoaffinity chromatography using antibodies specific for TMAH. Animmunoaffinity column is constructed by covalently coupling TMAHantibody to an activated chromatographic resin such as CnBr-activatedSepharose (Pharmacia Biotech). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Membrane fractions from cells expressing TMAH are prepared by methodswell known in the art. Alternatively, a recombinant TMAH fragmentcontaining an appropriate signal sequence may be secreted in usefulquantity into the medium in which transfected cells are grown.

A TMAH-containing preparation is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of TMAH (eg, high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/TMAH binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope such as urea or thiocyanate ion), and TMAH is collected.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 272 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: MPHGNOT03                                                        (B) CLONE: 513418                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetSerGluGluValThrTyrAlaAspLeuGlnPheGlnAsnSerSer                              151015                                                                        GluMetGluLysIleProGluIleGlyLysPheGlyGluLysAlaPro                              202530                                                                        ProAlaProSerHisValTrpArgProAlaAlaLeuPheLeuThrLeu                              354045                                                                        LeuCysLeuLeuLeuLeuIleGlyLeuGlyValLeuAlaSerMetPhe                              505560                                                                        HisValThrLeuLysIleGluMetLysLysMetAsnLysLeuGlnAsn                              65707580                                                                      IleSerGluGluLeuGlnArgAsnIleSerLeuGlnLeuMetSerAsn                              859095                                                                        MetAsnIleSerAsnLysIleArgAsnLeuSerThrThrLeuGlnThr                              100105110                                                                     IleAlaThrLysLeuCysArgGluLeuTyrSerLysGluGlnGluHis                              115120125                                                                     LysCysLysProCysProArgArgTrpIleTrpHisLysAspSerCys                              130135140                                                                     TyrPheLeuSerAspAspValGlnThrTrpGlnGluSerLysMetAla                              145150155160                                                                  CysAlaAlaGlnAsnAlaSerLeuLeuLysIleAsnAsnLysAsnAla                              165170175                                                                     LeuGluPheIleLysSerGlnSerArgSerTyrAspTyrTrpLeuGly                              180185190                                                                     LeuSerProGluGluAspSerThrArgGlyMetArgValAspAsnIle                              195200205                                                                     IleAsnSerSerAlaTrpValIleArgAsnAlaProAspLeuAsnAsn                              210215220                                                                     MetTyrCysGlyTyrIleAsnArgLeuTyrValGlnTyrTyrHisCys                              225230235240                                                                  ThrTyrLysLysArgMetIleCysGluLysMetAlaAsnProValGln                              245250255                                                                     LeuValLeuHisIleLeuGlyArgHisGluAlaSerIleLysTyrIle                              260265270                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 970 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: MPHGNOT03                                                        (B) CLONE: 513418                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GAAGTGTAAACTTGTAAGCTTAAGCTTCCGTTTATAAACAGAAGTTTAAAATTATAGGTC60                CTGTTTAACATTCAGCTCTGTTAACTCACTCATCTTTTTGTGTTTTTACACTTTGTCAAG120               ATTTCTTTACATATTCATCAATGTCTGAAGAAGTTACTTATGCAGATCTTCAATTCCAGA180               ACTCCAGTGAGATGGAAAAAATCCCAGAAATTGGCAAATTTGGGGAAAAAGCACCTCCAG240               CTCCCTCTCATGTATGGCGTCCAGCAGCCTTGTTTCTGACTCTTCTGTGCCTTCTGTTGC300               TCATTGGATTGGGAGTCTTGGCAAGCATGTTTCATGTAACTTTGAAGATAGAAATGAAAA360               AAATGAACAAACTACAAAACATCAGTGAAGAGCTCCAGAGAAATATTTCTCTACAACTGA420               TGAGTAACATGAATATCTCCAACAAGATCAGGAACCTCTCCACCACACTGCAAACAATAG480               CCACCAAATTATGTCGTGAGCTATATAGCAAAGAACAAGAGCACAAATGTAAGCCTTGTC540               CAAGGAGATGGATTTGGCATAAGGACAGCTGTTATTTCCTAAGTGATGATGTCCAAACAT600               GGCAGGAGAGTAAAATGGCCTGTGCTGCTCAGAATGCCAGCCTGTTGAAGATAAACAACA660               AAAATGCATTGGAATTTATAAAATCCCAGAGTAGATCATATGACTATTGGCTGGGATTAT720               CTCCTGAAGAAGATTCCACTCGTGGTATGAGAGTGGATAATATAATCAACTCCTCTGCCT780               GGGTTATAAGAAACGCACCTGACTTAAATAACATGTATTGTGGATATATAAATAGACTAT840               ATGTTCAATATTATCACTGCACTTATAAAAAAAGAATGATATGTGAGAAGATGGCCAATC900               CAGTGCAGTTGGTTCTACATATTTTAGGGAGGCATGAGGCATCAATCAAATACATTTAAG960               GAGTGTAGGG970                                                                 (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 172 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 293274                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetAlaGluSerGluLeuMetHisIleHisSerLeuAlaGluHisTyr                              151015                                                                        LeuGlnTyrValLeuGlnValProAlaPheGluSerAlaProSerGln                              202530                                                                        AlaCysArgValLeuGlnArgValAlaPheSerValGlnLysGluVal                              354045                                                                        GluLysAsnLeuLysSerTyrLeuAspAspPheHisValGluSerIle                              505560                                                                        AspThrAlaArgIleIlePheAsnGlnValMetGluLysGluPheGlu                              65707580                                                                      AspGlyIleIleAsnTrpGlyArgIleValThrIlePheAlaPheGly                              859095                                                                        GlyValLeuLeuLysLysLeuProGlnGluGlnIleAlaLeuAspVal                              100105110                                                                     CysAlaTyrLysGlnValSerSerPheValAlaGluPheIleMetAsn                              115120125                                                                     AsnThrGlyGluTrpIleArgGlnAsnGlyGlyTrpGluAspGlyPhe                              130135140                                                                     IleLysLysPheGluProLysSerGlyTrpLeuThrPheLeuGlnMet                              145150155160                                                                  ThrGlyGlnIleTrpGluMetLeuPheLeuLeuLys                                          165170                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 239 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 179367                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaHisAlaGlyArgThrGlyTyrAspAsnArgGluIleValMet                              151015                                                                        LysTyrIleHisTyrLysLeuSerGlnArgGlyTyrGluTrpAspAla                              202530                                                                        GlyAspValGlyAlaAlaProProGlyAlaAlaProAlaProGlyIle                              354045                                                                        PheSerSerGlnProGlyHisThrProHisProAlaAlaSerArgAsp                              505560                                                                        ProValAlaArgThrSerProLeuGlnThrProAlaAlaProGlyAla                              65707580                                                                      AlaAlaGlyProAlaLeuSerProValProProValValHisLeuAla                              859095                                                                        LeuArgGlnAlaGlyAspAspPheSerArgArgTyrArgGlyAspPhe                              100105110                                                                     AlaGluMetSerSerGlnLeuHisLeuThrProPheThrAlaArgGly                              115120125                                                                     ArgPheAlaThrValValGluGluLeuPheArgAspGlyValAsnTrp                              130135140                                                                     GlyArgIleValAlaPhePheGluPheGlyGlyValMetCysValGlu                              145150155160                                                                  SerValAsnArgGluMetSerProLeuValAspAsnIleAlaLeuTrp                              165170175                                                                     MetThrGluTyrLeuAsnArgHisLeuHisThrTrpIleGlnAspAsn                              180185190                                                                     GlyGlyTrpAspAlaPheValGluLeuTyrGlyProSerMetArgPro                              195200205                                                                     LeuPheAspPheSerTrpLeuSerLeuLysThrLeuLeuSerLeuAla                              210215220                                                                     LeuValGlyAlaCysIleThrLeuGlyAlaTyrLeuSerHisLys                                 225230235                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 197 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 34678                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetAlaProLysLysProGluProLysLysGluAlaAlaLysProMet                              151015                                                                        AsnValLysMetLeuAspPheGluThrPheLeuProIleLeuAlaPro                              202530                                                                        AlaProAlaProAlaProAlaProAlaProAlaProAlaGlnHisIle                              354045                                                                        SerArgAsnLysGluGlnGlyThrTyrGluAspPheProGluAlaPro                              505560                                                                        LysGluProAlaPheAspProLysSerValLysValGluGlyLeuArg                              65707580                                                                      ValPheAspLysGluSerAsnGlyThrValIleAspPheThrAlaAsp                              859095                                                                        GlnIleGluGluPheLysGluAlaPheMetGlyAlaGluLeuArgHis                              100105110                                                                     ValLeuAlaThrLeuGlyGluLysSerLeuPheAspArgThrProThr                              115120125                                                                     GlyGluMetLysIleThrTyrMetThrGluAlaGluValGluGlnLeu                              130135140                                                                     LeuAlaGlyGlnGluAspGlyGlnCysGlyAspValLeuArgAlaLeu                              145150155160                                                                  GlyGlnAsnProThrAlaAsnGlyCysIleAsnTyrGluAlaPheVal                              165170175                                                                     LysHisIleMetAsnAlaGluValLeuArgValLeuGlyLysProLys                              180185190                                                                     ProGluGluSerGly                                                               195                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 231 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 35061                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetSerLysGlnArgGlyThrPheSerGluValSerLeuAlaGlnAsp                              151015                                                                        ProLysArgGlnGlnArgLysProLysGlyAsnLysSerSerIleSer                              202530                                                                        GlyThrGluGlnGluIlePheGlnValGluLeuAsnLeuGlnAsnPro                              354045                                                                        SerLeuAsnHisGlnGlyIleAspLysIleTyrAspCysGlnGlyLeu                              505560                                                                        LeuProProProGluLysLeuThrAlaGluValLeuGlyIleIleCys                              65707580                                                                      IleValLeuMetAlaThrValLeuLysThrIleValLeuIleProPhe                              859095                                                                        LeuGluGlnAsnAsnSerSerProAsnThrArgThrGlnLysAlaArg                              100105110                                                                     HisCysGlyHisCysProGluGluTrpIleThrTyrSerAsnSerCys                              115120125                                                                     TyrTyrIleGlyLysGluArgArgThrTrpGluGluSerLeuLeuAla                              130135140                                                                     CysThrSerLysAsnSerSerLeuLeuSerIleAspAsnGluGluGlu                              145150155160                                                                  IleLysPheLeuAlaSerIleLeuProSerSerTrpIleGlyValPhe                              165170175                                                                     ArgAsnSerSerHisHisProTrpValThrIleAsnGlyLeuAlaPhe                              180185190                                                                     LysHisLysIleLysAspSerAspAsnAlaGluLeuAsnCysAlaVal                              195200205                                                                     LeuGlnValAsnArgLeuLysSerAlaGlnCysGlySerSerMetIle                              210215220                                                                     TyrHisCysLysHisLysLeu                                                         225230                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 215 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 35059                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetAspAsnGlnGlyValIleTyrSerAspLeuAsnLeuProProAsn                              151015                                                                        ProLysArgGlnGlnArgLysProLysGlyAsnLysSerSerIleLeu                              202530                                                                        AlaThrGluGlnGluIleThrTyrAlaGluLeuAsnLeuGlnLysAla                              354045                                                                        SerGlnAspPheGlnGlyAsnAspLysThrTyrHisCysLysAspLeu                              505560                                                                        ProSerAlaProGluLysLeuIleValGlyIleLeuGlyIleIleCys                              65707580                                                                      LeuIleLeuMetAlaSerValValThrIleValValIleProSerArg                              859095                                                                        HisCysGlyHisCysProGluGluTrpIleThrTyrSerAsnSerCys                              100105110                                                                     TyrTyrIleGlyLysGluArgArgThrTrpGluGluSerLeuLeuAla                              115120125                                                                     CysThrSerLysAsnSerSerLeuLeuSerIleAspAsnGluGluGlu                              130135140                                                                     MetLysPheLeuSerIleIleSerProSerSerTrpIleGlyValPhe                              145150155160                                                                  ArgAsnSerSerHisHisProTrpValThrMetAsnGlyLeuAlaPhe                              165170175                                                                     LysHisGluIleLysAspSerAspAsnAlaGluLeuAsnCysAlaVal                              180185190                                                                     LeuGlnValAsnArgLeuLysSerAlaGlnCysGlySerSerIleIle                              195200205                                                                     TyrHisCysLysHisLysLeu                                                         210215                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 233 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 35057                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetAspAsnGlnGlyValIleTyrSerAspLeuAsnLeuProProAsn                              151015                                                                        ProLysArgGlnGlnArgLysProLysGlyAsnLysSerSerIleLeu                              202530                                                                        AlaThrGluGlnGluIleThrTyrAlaGluLeuAsnLeuGlnLysAla                              354045                                                                        SerGlnAspPheGlnGlyAsnAspLysThrTyrHisCysLysAspLeu                              505560                                                                        ProSerAlaProGluLysLeuIleValGlyIleLeuGlyIleIleCys                              65707580                                                                      LeuIleLeuMetAlaSerValValThrIleValValIleProSerThr                              859095                                                                        LeuIleGlnArgHisAsnAsnSerSerLeuAsnThrArgThrGlnLys                              100105110                                                                     AlaArgHisCysGlyHisCysProGluGluTrpIleThrTyrSerAsn                              115120125                                                                     SerCysTyrTyrIleGlyLysGluArgArgThrTrpGluGluSerLeu                              130135140                                                                     LeuAlaCysThrSerLysAsnSerSerLeuLeuSerIleAspAsnGlu                              145150155160                                                                  GluGluMetLysPheLeuSerIleIleSerProSerSerTrpIleGly                              165170175                                                                     ValPheArgAsnSerSerHisHisProTrpValThrMetAsnGlyLeu                              180185190                                                                     AlaPheLysHisGluIleLysAspSerAspAsnAlaGluLeuAsnCys                              195200205                                                                     AlaValLeuGlnValAsnArgLeuLysSerAlaGlnCysGlySerSer                              210215220                                                                     IleIleTyrHisCysLysHisLysLeu                                                   225230                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 179 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 1098617                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetAlaValPheLysThrThrLeuTrpArgLeuIleSerGlyThrLeu                              151015                                                                        GlyIleIleCysLeuSerLeuMetAlaThrLeuGlyIleLeuLeuLys                              202530                                                                        AsnSerPheThrLysLeuSerIleGluProAlaPheThrProGlyPro                              354045                                                                        AsnIleGluLeuGlnLysAspSerAspCysCysSerCysGlnGluLys                              505560                                                                        TrpValGlyTyrArgCysAsnCysTyrPheIleSerSerGluGlnLys                              65707580                                                                      ThrTrpAsnGluSerArgHisLeuCysAlaSerGlnLysSerSerLeu                              859095                                                                        LeuGlnLeuGlnAsnThrAspGluLeuAspPheMetSerSerSerGln                              100105110                                                                     GlnPheTyrTrpIleGlyLeuSerTyrSerGluGluHisThrAlaTrp                              115120125                                                                     LeuTrpGluAsnGlySerAlaLeuSerGlnTyrLeuPheProSerPhe                              130135140                                                                     GluThrPheAsnThrLysAsnCysIleAlaTyrAsnProAsnGlyAsn                              145150155160                                                                  AlaLeuAspGluSerCysGluAspLysAsnArgTyrIleCysLysGln                              165170175                                                                     GlnLeuIle                                                                     __________________________________________________________________________

We claim:
 1. An isolated and purified polynucleotide sequence encodingthe polypeptide comprising the amino acid sequence of SEQ ID NO:1. 2.The polynucleotide sequence of claim 1 consisting of the nucleic acidsequence of SEQ ID NO:2.
 3. A polynucleotide sequence consisting of thecomplement of the nucleic acid sequence of claim
 2. 4. An isolated andpurified polynucleotide sequence that hybridizes under stringentconditions to the nucleic acid sequence of SEQ ID NO:2.
 5. A recombinantexpression vector containing the polynucleotide sequence of claim
 2. 6.A recombinant host cell containing the polynucleotide sequence of claim2.
 7. A method for producing a polypeptide comprising the amino acidsequence of SEQ ID NO:1, the method comprising the steps of:a) culturingthe host cell of claim 6 under conditions suitable for the expression ofthe polypeptide; and b) recovering the polypeptide from the host cellculture.